The present invention relates, specifically, to the transformation of maize X112 mutant ahas 2 gene into monocots such as maize (corn), wheat and rice, selection of transformed maize (corn), rice and wheat cells with imidazolinone, production of transgenic maize (corn), rice and wheat materials and plants resistant to the imidazolinone herbicides, in vitro characterization of the transformed plants, and greenhouse performances of imidazolinone resistant transgenic maize (corn), rice and wheat plants treated with various herbicides.
The existence of the branch chain amino acid (valine, leucine and isoleucine) biosynthetic pathway in plants, and its absence in animals is one of the major differences of plant and animal biochemistry. Therefore, inhibition of the branch chain amino acid biosynthesis is detrimental to plants but has no effect on animals. Imidazolinone and sulfonylurea herbicides inhibit, acetohydroxyacid synthase (AHAS, or acetolactate synthasexe2x80x94ALS; E.C.4.1.3.18), the key enzyme in the biosynthesis of branch chain amino acids (Chaleff and Mauvais, 1984; Shaner et al. 1984). Consequently, because imidazolinone and sulfonylurea herbicides are effective at very low application rates, and relatively non-toxic to animals, they are widely used in modern agriculture.
The differential sensitivity to the imidazolinone herbicides is dependent on the chemical nature of the particular herbicide and differential metabolism of the compound from toxic to non-toxic form in the plants (Shaner et al. 1984; Brown et al. 1987). Other plant physiological differences such as absorption and translocation also play an important role in selectivity (Shaner and Robinson 1985). Computer-based modeling of the three dimensional conformation of the AHAS-inhibitor complex predicts several amino acids in the proposed inhibitor binding pocket as sites where introduced mutations would likely confer selective resistance to imidazolinones (Ott et al. 1996). Transgenic plants produced with these rationally designed mutations in the proposed biding sites of the AHAS enzyme exhibited specific resistance to a single class of herbicides (Ott et al. 1996).
Application of imidazolinone herbicides in field production of major crops enables more effective weed control and less environmental impact than other chemicals. Among the major agricultural crops, only soybean is naturally resistant to imidazolinone herbicides due to its ability to rapidly metabolize the compounds (Shaner and Robinson 1985) while others such as corn (Newhouse et al. 1991), wheat (Newhouse et al. 1992) and rice (Barrette et al. 1989) are somewhat susceptible. In order to extend the application of imidazolinone and sulfonylurea herbicides to more crops, it is necessary to enhance plant resistance to these compounds. To date, three major approaches have been used to enhance plant resistance: 1) screening for spontaneous resistant mutations in cell culture (Chaleff and Ray 1984; Anderson and Georgeson 1989; Sebastian et al. 1989; Magha et al. 1993), 2) artificially inducing mutations in seeds or microspores (Swanson et al. 1989; Newhouse et al. 1992; Croughan 1996), and 3) transferring resistance genes between different species by genetic engineering (Haughn et al. 1988; Charest et al. 1990; Odell et al. 1990; Li et al. 1992; Tourneur et al. 1993). Thus far spontaneous herbicide resistant mutants have been identified and characterized in tobacco (Chaleff and Ray 1984), soybean (Sebastian et al. 1989), corn (Anderson and Georgeson 1989) and rapeseed (Magha et al. 1993). Chemical mutagenesis successfully produced resistant mutants in wheat (Newhouse et al. 1992), canola (Swanson et al. 1989) and rice (Croughan 1996). Studies on tobacco (Haughn et al. 1988; Odell et al. 1990; Charest et al. 1990) and rice (Li et al. 1992) suggested the potential of transferring genes from one species to another for the production of resistant crops.
Advances in transformation technologies of monocots, especially of rice have made possible the transfer of genes between species for development of transgenic plants with improved characteristics. Transgenic rice plants have been produced by transformation of protoplasts (Shimamoto et al. 1989; Peng et al. 1990), bombardment of cells (Christou et al. 1991; Li et al. 1993), and more recently, Agrobacterium-mediated transformation of immature embryos (Chan et al. 1992; Hiei et al. 1994; Aldemita et al. 1996). Critical in all the transformation processes is the ability to select for the cells that have been transformed over the rest of the population of cells. Typically a combination of an antibiotic and a gene conferring resistance to the antibiotic has been used. Examples include the neomycin phosphotransferase (neo) gene for resistance to kanamycin or genetic (G-418), hygromycin B transferase (hyh) for hygromycin B resistance (Shimamoto et al. 1989; Hayashimoto et al. 1990), and the bar gene for phophinothricin resistance (Christou et al. 1991; Rathore et al. 1993). All of these selectable genes (neo, hyh and bar) are of bacterial origin. In one report, use of a mutant als gene from Arabidopsis coupled with selection on sulfonylurea herbicide was demonstrated for production of transgenic rice plants (Li et al. 1992). An increase in in vitro resistance to chlorsulfuron of similar magnitude (200-fold) was demonstrated in transgenic rice containing 35S/als transgene (Li et al. 1992), and imidazolinone-resistant growth of transgenic tobacco was reported to be 100-fold greater than nontransformed control plants (Sathasivan et al.1991). In the literature, expression of the introduced AHAS (or ALS) gene at different magnitudes was achieved by manipulating several aspects of the transformation that included the use of different promoters and screening larger populations of transformants (Odell et al. 1990; Sathasivan et al. 1991; Li et al. 1992). Studies showed that replacing the Arabidopsis ALS promoter with the CaMV35S promoter resulted in 40-fold differences in in vitro resistance to chlorsulfuron (Li et al. 1992). In tobacco, increase in resistance to imazethapyr in individual calli transformed with mutant als gene from Arabidopsis ranged from 10- to 1000-fold, most likely reflecting the differences in gene copy numbers or in chromosomal positions of the transgenes (Sathasivan et al. 1991).
Imidazolinone-specific resistance has been reported in a number of patents. U.S. Pat. No. 4,761,373 described in general terms an altered ahas as a basis of herbicide resistance in plants, and specifically disclosed certain imidazolinone resistant corn lines. U.S. Pat. No. 5,013,659 disclosed that mutants exhibiting herbicide resistance possess mutations in at least one amino acid in one or more conserved regions. The mutations described therein encode either cross-resistance for imidazolinones and sulfonylureas or sulfonylurea-specific resistance but no imidazolinone-specific resistance. Additionally, U.S. Pat. No. 5,731,180 and continuation-in-part U.S. Pat. No. 5,767,361 isolated a gene encoding imidazolinone-specific resistance in a monocot and determined it to be associated with a single amino acid substitution in a wild-type moncot AHAS amino acid sequence. U.S. Pat. Nos. 5,731,180 and 5,767,361, as well as U.S. Pat. Nos. 5,750,866 and 6,025,541, are incorporated herein by reference. However, while the referenced patents generally allude to the use of the gene as a selectable marker for selection on imidazolinone, the present invention describes the specific application of the maize X112 mutant ahas 2 gene to monocots such as maize (corn), rice and wheat varieties, or use of the mutant XI12 ahas 2 gene as a selectable marker coupled with a imidazolinone compound as a selection system for resistance to the imidazolinone herbicides.
The AHAS gene codes for acetohydroxyacid synthase (AHAS, E.C.4.1.3.18; also called acetolactate synthase; ALS) which is the first common enzyme in the biosynthetic pathway of branch chain amino acids (Shaner et al. 1984). The imidazolinone herbicides are a class of herbicides that inhibit AHAS activity thus, preventing further growth and development of susceptible plants such as rice and many weed species. In biochemical studies, selectivity of the imidazolinone herbicides has been shown to be based on differences in nature and rate of metabolism of the herbicides (Shaner and Robinson 1985; Brown et al. 1987). In genetic studies, mutations in the ahas gene have been linked for resistance to the imidazolinone herbicides in canola (Swanson et al. 1989) and corn (Newhouse et al. 1991). Analysis of the mutant ahas 2 gene, isolated from maize (XI12) plants revealed that a single base mutation from G to A at nucleotide 621 relative to the initiation codon resulted in a one amino acid change in the AHAS enzyme from Ser to Asn (Dietrich, 1998). The term mutant ahas 2 gene as used herein includes any additions, deletions, or substitutions in the nucleic acid sequence as described in Dietrich, U.S. Pat. No. 5,731,180, that do not change the function of the mutant ahas 2 gene. The function of the mutant ahas 2 gene is to confer resistance to imidazolinone herbicides. The maize XI12 mutant ahas 2 gene presents a number of advantages for use in plant transformation. It is a plant gene and even this mutant form is known to exist in plant populations. A combination of the maize XI12 mutant ahas 2 gene and imidazolinone chemistry provides a useful system for selection of transformed cells, plants and progeny. Use of a plant mutant ahas gene for selection in plant transformation circumvents the problems associated with transformation of antibiotic gene of bacterial origin in transgenic plants. The term mutant ahas 2 gene as used herein includes any additions, deletions, or substitutions in the nucleic acid sequence as described in Dietrich, U.S. Pat. No. 5,731,180, that do not change the function of the mutant ahas 2 gene. The function of the mutant ahas 2 gene is to confer resistance to imidazolinone herbicides.
In this invention we report the transformation of maize X112 mutant ahas 2 gene into maize and wheat embryos and rice protoplasts, selection of transformed cells with an imidazolinone compound, production of transgenic maize, rice and wheat plants resistant to the imidazolinone herbicides, in vitro characterization of the transformed plants, and greenhouse performances of imidazolinone resistant transgenic plants treated with various herbicides.
In rice, resistant calli were recovered from transformations following selection on imazethapyr and regenerated into fertile plants. Genetic study on two transgenic rice lines (26 and 29) showed that the introduced maize ahas2 gene was stably transmitted to progeny plants and conferred a single dominant trait inherited in a Mendelian fashion. Homozygous imidazolinone resistant lines were readily identified and isolated based on seed germination and greenhouse screen tests. A 100-fold increase in in vitro resistance to imazethapyr in cell lines and immature embryos derived from transgenic homozygous resistant plants was demonstrated. Also the invention can be used to deliver a second gene in co-transformation. Also, the invention identifies the production of transgenic maize and wheat plants by Agrobacterium-mediated transformation using the maize X112 mutant ahas2 gene as a selectable marker coupled with selection of callus material and regeneration of plants on media supplemented with the imidazolinone herbicides. Transformation efficiency averaged about 2% and reached to as high as 16%-20% in some experiments for corn and ranged from 0.4 to 3.1% for wheat.
We evaluated performances of transgenic maize, rice and wheat plants containing the maize XI12 ahas 2 gene in response to applications of various herbicides in greenhouse. A total of 9 herbicides including five imidazolinones (PURSUIT(copyright) imazethapyr, CADRE(copyright) imazameth, RAPTOR(copyright) imazamox, SCEPTER(copyright) imazaquin, and ARSENAL(copyright) imazapyr, two rice herbicides, AC322,140 cyclosufamuron and LONDAX(copyright) bensulfuron-methyl, ACCENT(copyright) nicosulfuron and CLASSIC(copyright) clorimuron-ethyl applied at 4 different rate were used in a study of transgenic rice plants. The results showed that transgenic plants were resistant to the five imidazolinone herbicides at rates as high as 6xc3x97 typical use rates and retained sensitivity to the sulfonylurea herbicides as compared to untransformed control plants. When treated with herbicides, transgenic plants produced yields comparable with their untreated counterparts. Untransformed control plants, however, had 10 to 20% higher yields than transgenic plants in the absence of herbicide treatments. All herbicides at all rates tested had no detrimental effects on seed set, except higher rates of ARSENAL(copyright) imazapyr caused severe sterility in transgenic plants. Enzyme assays provided evidence that the introduced XI12 maize ahas 2 gene conferred selective resistant AHAS enzyme in the transgenic plants. Evaluation of effects of different imidazoline herbicides on transgenic corn plants showed there was no injury up to 16xc3x97ARSENALO(copyright) imazapyr (384 g/ha), 8xc3x97PURSUIT(copyright) imazethapyr (500 g/ha), 4xc3x97CADRE(copyright) imazameth (800 g/ha). At 4xc3x97imazamox (160 g/ha), no or slight injury to the plants was observed. An increase of 5xc3x97 tolerance to imazamox was demonstrated in transgenic wheat plants. The mutant ahas2 gene can be used as an effective selectable marker in transformation, useful in selection for stacked gene traits, useful as a selectable marker in breeding or hybrid seed production, and useful as a quality control tool.